ASMS 2014 Analytical Core Directors Workshop. Presented by Chris Colangelo http://medicine.yale.edu/keck/proteomics/index.aspx

1. Controlling and Measuring Variation
in Sample Preparation and Data
Analysis in a Core Facility
Environment
Christopher Colangelo
Director of Protein Profiling
MS & Proteomics Resource
Yale University
June 17, 2014
Analytical Lab Managers Interest Group
ASMS – Baltimore, MD

2. Sources of Technical Variability in Quantitative LC-MS
Proteomics: Human Brain Tissue Sample Analysis
Piehowski et. al. J. Proteome Res. 2013; 12(5): 2128–2137

3. Step 1:
• Educate your users
– Provide them with Standard Operating Procedures
– Sample Handling Procedures
– Standard Digestion Protocols
• Educate yourself and your staff
– Training classes, Workshops, Literature, Books
• learn statistics (CV, ANOVA, Blocking, T-test)
– Journal Clubs (discuss good vs. bad research)

4. Define the Goal of the experiment
- For each project
- Clearly articulate a goal
- Develop methods to measure results
- Outline Sample design
- Get Statistical help at beginning!!!
- Discuss internal and external standards
- Measure and repeat

5. Simplify
A
B
C
D

6. Design, Measure, and Repeat
• Design Assays which can Measure the variability in
each step of your process
• Measure this variability in a controlled experiment
• Replicates are essential (technical, biological)

7. Example
Measuring Between Animal, Cage, Prep Date, and Condition
cage rat ID tissue frozen or unfrozen prep-date fractions
A
1
half cortex unfrozen Day 1 Subfraction 1 and Subfraction 2
half cortex frozen Day 1 Subfraction 1 and Subfraction 2
2
half cortex unfrozen Day 1 Subfraction 1 and Subfraction 2
half cortex frozen Day 1 Subfraction 1 and Subfraction 2
B
3
half cortex unfrozen Day 2 Subfraction 1 and Subfraction 2
half cortex frozen Day 2 Subfraction 1 and Subfraction 2
4
half cortex unfrozen Day 2 Subfraction 1 and Subfraction 2
half cortex frozen Day 2 Subfraction 1 and Subfraction 2
C
5
half cortex frozen Day 2 Subfraction 1 and Subfraction 2
half cortex frozen Day 3 Subfraction 1 and Subfraction 2
6
half cortex frozen Day 2 Subfraction 1 and Subfraction 2
half cortex frozen Day 3 Subfraction 1 and Subfraction 2
D
7
half cortex frozen Day 2 Subfraction 1 and Subfraction 2
half cortex frozen Day 3 Subfraction 1 and Subfraction 2
8
half cortex frozen Day 2 Subfraction 1 and Subfraction 2
half cortex frozen Day 3 Subfraction 1 and Subfraction 2

8. PCA plot for Subfraction 1 and Subfraction 2
SWATH data from 1700 proteins
Red – Day 1, Green – Day 2, Blue – Day 3
circles: Cage A, squares: Cage B, triangles: Cage C
Solid(Filled) Rat 1,3,5, Open (No fill) Rat 2,4,6
Subfraction 2Subfraction 1

9. SubFraction 1 Subfraction 2
red: day1
green: day2
blue: day3
Effect of preparation conditions on ALL proteins
Clustering for Subfraction 1 and Subfraction 2
SWATH data from 1700 proteins

10. Day1
Day2
Day 0
PCA on Subfraction 1 (between animals, cages, or prep-dates)

11. red: day1
green: day2
blue: day3
Comparison of Subfraction 1 fractions between prep-dates

12. red: day1
green: day2
blue: day3
PCA on Subfraction 2 between prep-dates

13. Four steps
1. Educate
1. Define Goal
2. Simplify
3. Design, Measure
and Repeat
A B C